Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
The main problem facing plant molecular biologists and plant breeders today is how to identify agriculturally important genes that are known only by their phenotype. Although map-based cloning and transposon tagging have made it possible to identify plant genomic clones by observing a phenotype, it is a technically difficult and labor intensive process. To date only a few major plant genes of this type have been cloned (Martin et al. 1993, Bent et al. 1994, Jones et al. 1994, Whitham et al. 1994).
Plant breeders have always relied on genetic variation for classical breeding programs. To be most effective, modern plant breeders need to know the molecular biology of genes that are important to agriculture. If the gene coding for a desired trait has been cloned, it is now possible, in some instances, to bypass classical breeding techniques and to directly introduce that trait by plant transformation. In the future it may even be possible to introduce desirable traits from species that are not cross-compatible, something that cannot be achieved by classical plant breeding. The cloning of agriculturally important genes will also open up new areas of research in plant biology.
When the idea of map-based cloning was first envisioned, a chromosome walk to link up overlapping genomic clones in the region of interest was considered a necessity. Crop plants tend to have relatively large genomes and high levels of repetitive DNA, features that make chromosome walking more tedious and more complicated. Fortunately, the recent development of methods that make it possible to identify molecular markers closely linked to a gene have led to the concept of chromosome landing.
Chromosome landing is based on the fact that it is no longer that difficult to obtain markers that are closely linked to a gene of interest, even in a genome that is not well characterized. RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism) methods in combination with analysis of near isogenic lines or bulked segregants can be used to identify molecular markers that are very close to a gene of interest (Williams et al. 1990, Zabeau and Vos 1992, Martin et al. 1991, Michelmore et al. 1991). A genomic library can then be screened using markers that flank the gene to identify potential clones.
Currently, the most significant hurdle remaining for the wide scale application of map-based cloning is how to verify that a potential clone does indeed carry the gene of interest. Until now, the approach has been to generate a series of subclones of a yeast artificial chromosome (YAC) (Burke et al. 1987) or bacterial artificial chromosome (BAC) believed to contain a gene of interest. Each of these subclones must then be individually transformed into plants to identify the presence (or absence) of the gene by assaying transgenic plants for the expected phenotype. Not only is this very time consuming, it is a risky proposition since any given subclone may contain only. part of the target gene, and thus give a negative result in transformation/complementation experiments. If the target gene contains a large number of introns and thus stretches over a large segment of genomic DNA, then none of the subclones will be sufficiently large to contain the entire gene.
What is needed most is technology that can circumvent this bottleneck of gene verification. The ability to transfer large DNA constructs to plants would reduce the amount of subcloning required from a high molecular weight YAC or BAC library vector before proceeding on to plant transformation. A high molecular weight library vector that can be used to transform plants directly would be ideal. Indeed, the development of technology to transform plants with high molecular weight DNA will be essential for the cloning of QTLs (quantitative trait loci) which make up the major portion of agriculturally important genes.
None of the existing vectors for high molecular weight genomic libraries are suitable for plant transformation. A series of cosmid library vectors (maximum insert size 46 kb) suitable for Agrobacterium-mediated plant transformation were constructed by Ma et al. (1992). However, the small insert size makes them less practical for genomes much larger than Arabidopsis. Whereas Arabidopsis is an attractive model system because its genomic size is small, about 100 megabase-pairs (Mbp), other higher plants have 4-20-fold larger genomes (Bennett and Smith 1976). The first high molecular weight plant genomic libraries were constructed in YAC vectors. The Pto disease resistance gene from tomato (Martin et al. 1992) and the RPS2 disease resistance gene from Arabidopsis (Bent et al. 1994) have been cloned from YAC libraries. However, a YAC library is a major investment to construct, maintain and utilize, and some YAC libraries have been plagued by deletions, rearrangements, and chimeras (Anderson 1993).
Recently, BAC libraries have been constructed for sorghum (Woo et al. 1994) and for rice (Wang and Ronald 1994) with average insert sizes of about 180 kb. BAC libraries have proven to be easier to construct, screen and maintain than YAC libraries.
Agrobacterium tumefaciens is a naturally occurring plant transformation system (see generally, review by Kado 1991; and Zambryski 1988). Recently, Miranda et al. showed that at least 170 kb could be transferred from the Ti plasmid of Agrobacterium to a plant genome (Miranda et al. 1992). The most dramatic potential for Agrobacterium-mediated transformation of plants with high molecular weight constructs is that A. tumefaciens has been shown to transfer its T-DNA to the plant genome in a linear fashion. It would be a tremendous advantage if transgenic plants could be generated that carry the entire genomic library insert (intact) in the plant genome. Linear transfer would eliminate the possibility of truncated genes except at the limits of the inserts, and make it possible to introduce very large plant genes (if they exist), or gene clusters.
Agrobacterium-mediated plant transformation is the most widely used technique for introducing new genetic information into plant cells. Agrobacterium-mediated transformation is routinely applicable in species like tobacco, petunia, and tomato, and a considerable amount of effort has gone into applying this technique to major crop species (reviewed by van Wordragen and Dons 1992) and monocot species. Recently, Agrobacterium-mediated transformation of rice has been reported (Hiei et al. 1994).
A need continues to exist, however, for a vector to transform plants with high molecular weight DNA sequences.